American Society of Hematology

Figure 2

$Figure 2. KLRG1+/CX3CR1-GFP+ NK cells localize in the BM sinusoids and display reduced CXCR4 expression and CXCL12 responsiveness. (A) Immunofluorescence staining of BM cells with anti-NK1.1–, anti-CD3ϵ–, anti-KLRG1–, and anti-CXCR4–specific antibodies was performed, followed by FACS analysis. (Left panel) histogram overlays show CXCR4 geometric mean fluorescence intensity (MFI) of KLRG1+/CX3CR1-GFP− and KLRG1+/CX3CR1-GFP+ NK cells, of 1 representative experiment of at least 3 performed. (Right panel) histograms show the mean values ± SD of CXCR4 geometric MFI of KLRG1−, KLRG1+/CX3CR1-GFP− and KLRG1+/CX3CR1-GFP+ BM NK cells of a total of 8 animals analyzed in independent experiments. (B) Ex vivo chemotaxis assays with CXCL12 (200 ng/mL) were performed in 5 μm pore Transwell insert. Migrated cells were stained with anti-NK1.1–, anti-KLRG1–, and anti-CD3ϵ–specific mAbs and counted by FACS analysis. Histograms show migration of the indicated BM NK-cell subsets in response to migration medium (NT) or CXCL12. Data are expressed as percentage of input cells and represent the means ± SD of percentage of migrated cells from a total of 6 animals analyzed in independent experiments. Student t test was performed by comparing CXCR4 expression or CXCL12-supported migration of KLRG1−, vs KLRG1+/CX3CR1-GFP− and vs KLRG1+/CX3CR1-GFP+ NK cells; *P < .05. (C) BM sinusoidal NK cells were labeled by intravenous injection of 1 μg of DX5-PE mAb in 300 μL PBS. The percentage of DX5-PE+ cells within KLRG1−, KLRG1+/CX3CR1-GFP− and KLRG1+/CX3CR1-GFP+ NK cells was evaluated. Histogram plots (top panel) show a representative experiment of at least 3 performed. Histograms (bottom left panel) show the mean values ± SD of the percentage of DX5+ cells in the different BM NK-cell populations of a total of 9 animals analyzed in independent experiments. Histograms (bottom right panel) show the fraction of parenchymal (Par, DX5-PE−) and sinusoidal (Sin, DX5-PE+) NK cells of the indicated phenotypes within total NK cells of the parenchyma and sinusoids, respectively. NK-cell number (×103) in parenchyma and sinusoids were, respectively, 173 ± 19 and 12.8 ± 7.3 for KLRG1−; 10.1 ± 2.0 and 4.3 ± 1.3 for KLRG1+/CX3CR1-GFP−; 2.5 ± 0.8 and 4.1 ± 2.7 for KLRG1+/CX3CR1-GFP+. (D) CX3CR1+/GFP mice were injected subcutaneously with 50 μL PBS alone (−) or containing 2 mg/mL AMD-3100, and sinusoidal NK cells were stained by intravenous administration of DX5-PE mAb in the last 2 minutes of treatment. Mice were killed and BM cells were counted and stained with anti-CD3ϵ–, anti-NK1.1–, anti-KLRG1–specific mAbs. Histograms represent the mean values ± SD of total cell number of the indicated NK-cell subsets in BM parenchyma (left panel) and sinusoids (right panel) from a total of 6 animals/group analyzed in independent experiments. Student t test was performed to compare the BM distribution of NK-cell subsets in mice treated with vehicle control with that of mice treated with AMD-3100. *P < .05.$

KLRG1⁺/CX3CR1-GFP⁺ NK cells localize in the BM sinusoids and display reduced CXCR4 expression and CXCL12 responsiveness. (A) Immunofluorescence staining of BM cells with anti-NK1.1–, anti-CD3ϵ–, anti-KLRG1–, and anti-CXCR4–specific antibodies was performed, followed by FACS analysis. (Left panel) histogram overlays show CXCR4 geometric mean fluorescence intensity (MFI) of KLRG1⁺/CX3CR1-GFP⁻ and KLRG1⁺/CX3CR1-GFP⁺ NK cells, of 1 representative experiment of at least 3 performed. (Right panel) histograms show the mean values ± SD of CXCR4 geometric MFI of KLRG1⁻, KLRG1⁺/CX3CR1-GFP⁻ and KLRG1⁺/CX3CR1-GFP⁺ BM NK cells of a total of 8 animals analyzed in independent experiments. (B) Ex vivo chemotaxis assays with CXCL12 (200 ng/mL) were performed in 5 μm pore Transwell insert. Migrated cells were stained with anti-NK1.1–, anti-KLRG1–, and anti-CD3ϵ–specific mAbs and counted by FACS analysis. Histograms show migration of the indicated BM NK-cell subsets in response to migration medium (NT) or CXCL12. Data are expressed as percentage of input cells and represent the means ± SD of percentage of migrated cells from a total of 6 animals analyzed in independent experiments. Student t test was performed by comparing CXCR4 expression or CXCL12-supported migration of KLRG1⁻, vs KLRG1⁺/CX3CR1-GFP⁻ and vs KLRG1⁺/CX3CR1-GFP⁺ NK cells; *P < .05. (C) BM sinusoidal NK cells were labeled by intravenous injection of 1 μg of DX5-PE mAb in 300 μL PBS. The percentage of DX5-PE⁺ cells within KLRG1⁻, KLRG1⁺/CX3CR1-GFP⁻ and KLRG1⁺/CX3CR1-GFP⁺ NK cells was evaluated. Histogram plots (top panel) show a representative experiment of at least 3 performed. Histograms (bottom left panel) show the mean values ± SD of the percentage of DX5⁺ cells in the different BM NK-cell populations of a total of 9 animals analyzed in independent experiments. Histograms (bottom right panel) show the fraction of parenchymal (Par, DX5-PE⁻) and sinusoidal (Sin, DX5-PE⁺) NK cells of the indicated phenotypes within total NK cells of the parenchyma and sinusoids, respectively. NK-cell number (×10³) in parenchyma and sinusoids were, respectively, 173 ± 19 and 12.8 ± 7.3 for KLRG1⁻; 10.1 ± 2.0 and 4.3 ± 1.3 for KLRG1⁺/CX3CR1-GFP⁻; 2.5 ± 0.8 and 4.1 ± 2.7 for KLRG1⁺/CX3CR1-GFP⁺. (D) CX3CR1^+/GFP mice were injected subcutaneously with 50 μL PBS alone (−) or containing 2 mg/mL AMD-3100, and sinusoidal NK cells were stained by intravenous administration of DX5-PE mAb in the last 2 minutes of treatment. Mice were killed and BM cells were counted and stained with anti-CD3ϵ–, anti-NK1.1–, anti-KLRG1–specific mAbs. Histograms represent the mean values ± SD of total cell number of the indicated NK-cell subsets in BM parenchyma (left panel) and sinusoids (right panel) from a total of 6 animals/group analyzed in independent experiments. Student t test was performed to compare the BM distribution of NK-cell subsets in mice treated with vehicle control with that of mice treated with AMD-3100. *P < .05.

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